Novel 2-substituted-benzimidazole-6-sulfonamides as carbonic anhydrase inhibitors: synthesis, biological evaluation against isoforms I, II, IX and XII and molecular docking studies

Article abstract of DOI:10.1080/14756366.2019.1666836

Inhibition of Carbonic Anhydrases (CAs) has been clinically exploited for many decades for a variety of therapeutic applications. Within a research project aimed at developing novel classes of CA inhibitors (CAIs) with a proper selectivity for certain iso

Full text of DOI:10.1080/14756366.2019.1666836

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Novel 2-substituted-benzimidazole-6-sulfonamides
as carbonic anhydrase inhibitors: synthesis,
biological evaluation against isoforms I, II, IX and
XII and molecular docking studies
Ciro Milite, Giorgio Amendola, Alessio Nocentini, Silvia Bua, Alessandra
Cipriano, Elisabetta Barresi, Alessandra Feoli, Ettore Novellino, Federico Da
Settimo, Claudiu T. Supuran, Sabrina Castellano, Sandro Cosconati & Sabrina
Taliani
To cite this article: Ciro Milite, Giorgio Amendola, Alessio Nocentini, Silvia Bua, Alessandra
Cipriano, Elisabetta Barresi, Alessandra Feoli, Ettore Novellino, Federico Da Settimo, Claudiu T.
Supuran, Sabrina Castellano, Sandro Cosconati & Sabrina Taliani (2019) Novel 2-substituted-
benzimidazole-6-sulfonamides as carbonic anhydrase inhibitors: synthesis, biological evaluation
against isoforms I, II, IX and XII and molecular docking studies, Journal of Enzyme Inhibition and
Medicinal Chemistry, 34:1, 1697-1710, DOI: 10.1080/14756366.2019.1666836
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2019, VOL. 34, NO. 1, 1697–1710
https://doi.org/10.1080/14756366.2019.1666836
RESEARCH PAPER
Novel 2-substituted-benzimidazole-6-sulfonamides as carbonic anhydrase
inhibitors: synthesis, biological evaluation against isoforms I, II, IX and XII and
molecular docking studies
a
, Giorgio Amendolab
, Alessio Nocentinic , Silvia Buac , Alessandra Ciprianoa,d
,
ꢀ
ꢀ
Ciro Milite
Elisabetta Barresie , Alessandra Feolia , Ettore Novellinof , Federico Da Settimoe , Claudiu T. Supuranc
,
Sabrina Castellanoa , Sandro Cosconatib
and Sabrina Talianie
a
b
ꢀ
Department of Pharmacy, Epigenetic Med Chem Lab, University of Salerno, Fisciano (SA), Italy; DiSTABiF, Universita della Campania Luigi
c
ꢀ
Vanvitelli, Caserta, Italy; NEUROFARBA Department, Sezione di Scienze Farmaceutiche e Nutraceutiche, Universita degli Studi di Firenze, Sesto
Fiorentino (Florence), Italy; PhD Program in Drug Discovery and Development, University of Salerno, Fisciano (SA), Italy; Department of
Pharmacy, Universy of Pisa, Pisa, Italy; Department of Pharmacy, University Federico II of Naples, Naples, Italy
d
e
f
ABSTRACT
ARTICLE HISTORY
Received 26 July 2019
Revised 5 September 2019
Accepted 6 September 2019
Inhibition of Carbonic Anhydrases (CAs) has been clinically exploited for many decades for a variety of
therapeutic applications. Within a research project aimed at developing novel classes of CA inhibitors
(CAIs) with a proper selectivity for certain isoforms, a series of derivatives featuring the 2-substituted-
benzimidazole-6-sulfonamide scaffold, conceived as frozen analogs of Schiff bases and secondary amines
previously reported in the literature as CAIs, were investigated. Enzyme inhibition assays on physiologically
relevant human CA I, II, IX and XII isoforms revealed a number of potent CAIs, showing promising selectiv-
ity profiles towards the transmembrane tumor-associated CA IX and XII enzymes. Computational studies
were attained to clarify the structural determinants behind the activities and selectivity profiles of the
novel inhibitors.
KEYWORDS
Carbonic anhydrase
inhibitors; benzimidazole-
sulfonamides; reduced
flexibility approach;
isoform-selective inhibitors;
molecular docking
GRAPHICAL ABSTRACT
Introduction
VA and CA VB), and secreted in saliva (CA VI) enzymes were char-
acterized1. Most of these CA isoforms represent interesting thera-
peutic targets, and their inhibition has been exploited clinically for
many decades for a variety of applications in treating a multitude
of diseases such as glaucoma, edema, epilepsy, obesity, neuro-
pathic pain and other neurological disorders2–4. More recently,
hCA IX and XII have been implicated in tumor progression/metas-
tasis, and their selective inhibition could represent an additional
opportunity for drug intervention against hypoxic cancers5.
Carbonic anhydrases (CA) are a family of ubiquitary zinc metal-
loenzymes that catalyze the reversible reaction of hydration of
CO2 to HCO3ꢁ 1. This simple transformation plays a physiological
regulatory role in a number of processes associated with pH con-
trol, ion transport, fluid secretion and several biosynthetic path-
ways1. Fifteen CA isoenzymes are encoded in humans and other
primates, that differ for their subcellular localization, catalytic
activity, and susceptibility to different classes of inhibitors1.
Specifically, cytosolic (CA I, CA II, CA III, CA VII, and CA XIII), mem-
brane-bound (CA IV, CA IX, CA XII and CA XIV), mitochondrial (CA
Sulfonamides and their bioisosteres (sulfamates, sulfamides) are
known, powerful CA inhibitors (CAIs)6. Acetazolamide 1 (AAZ),
CONTACT Sabrina Castellano
scastellano@unisa.it
Department of Pharmacy, Epigenetic Med Chem Lab, University of Salerno, via Giovanni Paolo II 132, I-
84084 Fisciano, Salerno, Italy; Sandro Cosconati
Caserta, Italy
sandro.cosconati@unicampania.it DiSTABiF, University of Campania “Luigi Vanvitelli”, Via Vivaldi 43, 81100
ꢀ
These two authors equally contributed to this paper.
Supplemental data for this article can be accessed here.
ß 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

1698
C. MILITE ET AL.
Chart 1. Structures of clinically used CAIs.
Figure 1. Flowchart of our frozen analog approach.
methazolamide 2, ethoxzolamide 3, brinzolamide 4 and dorzola- giving interactions with the enzyme that may not involve a direct
mide 5 are among the CAIs used in medicine mainly as diuretics interaction with the active site zing ion15–17. Herein, we report the
and antiglaucoma agents (Chart 1)6.
synthesis of 2-substituted-benzimidazole-6-sulfonamides 6–18 and
Because of the ubiquity of CAs, the selectivity of the inhibitors
for certain isoforms is a crucial issue to be reached in a drug
development campaign in order to target a disease without rele-
vant side effects7. In this respect, expanding the chemical space
by the exploration of novel scaffolds may aid the development of
novel classes of CAIs featuring improved pharmacological proper-
ties in terms of inhibition potency and isoform-selectivity.
the related carboxamide 19 and their inhibitory activity toward
four physiologically relevant enzymes, the cytosolic isoforms hCA I
and II as well as the transmembrane tumor-associated ones hCA
IX and XII.
Materials and methods
Among the several sulfonamide CAIs described, Schiff bases
and secondary amines incorporating aromatic/heterocyclic sul-
fonamide moieties in their structure (compounds of type I in
Chemistry
All chemicals were purchased from Sigma Aldrich Srl (Milan, Italy)
or from Fluorochem Ltd. (Hadfield, UK) and were of the highest
purity. All solvents were reagent grade and, when necessary, were
purified and dried by standard methods. All reactions requiring
anhydrous conditions were conducted under a positive atmos-
phere of nitrogen in oven-dried glassware. Standard syringe tech-
niques were used for anhydrous addition of liquids. Reactions
were routinely monitored by TLC performed on aluminum-backed
silica gel plates (Merck DC, Alufolien Kieselgel 60 F254) with spots
visualized by UV light (k ¼ 254, 365 nm) or using a KMnO4 alkaline
solution. Solvents were removed using a rotary evaporator operat-
ing at a reduced pressure of ꢂ10 Torr. Organic solutions were
dried over anhydrous Na2SO4. Chromatographic purification was
done on an automated flash-chromatography system (IsoleraTM
Figure 1) have been extensively investigated in recent years8–11
.
Comparing the activity of imines and their secondary amine coun-
terparts, it is evident that the molecular flexibility markedly affects,
positively or negatively, both activity and selectivity8–11
.
Prompted by these outcomes and exploiting our experience in
the application of the frozen analog approach12,13, we decided to
further reduce the flexibility of the Schiff bases constraining the
N¼C imine bond into a ring (II, Figure 1). As a rigidifying building
block, we decided to introduce the benzimidazole (III, Figure 1), a
privileged structure extensively used in medicinal chemistry, which
has been only scarcely explored for its potential in the develop-
ment of CAIs14. As substituent in the position 2 of benzimidazole
core, we selected phenols and benzoic acid derivatives. In fact, dif-
ferent studies showed that these scaffolds are effective CAIs, Dalton 2000, Biotage) using cartridges packed with KP-SIL, 60 Å

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1699
(40–63 mm particle size). All microwave assisted reactions were 7.93 (s, 1H), 7.63–7.58 (m, 2H), 7.21 (s, 2H, exchangeable with
R
conducted in
equipped with
a
CEM DiscoverV SP microwave synthesizer D2O), 7.01 (d, J ¼ 8.1 Hz, 2H), 6.64 (d, J ¼ 8.1 Hz, 2H), 3.09 (t,
a
vertically focused IR temperature sensor. J ¼ 7.5 Hz, 2H), 2.99 (t, J ¼ 7.5 Hz, 2H). 13C NMR (100 MHz, DMSO) d
Analytical high performance liquid chromatography (HPLC) was
performed on a Shimadzu SPD 20 A UV/VIS detector (k ¼ 220 and
254 nm) using C-18 column Phenomenex Synergi Fusion - RP 80 A
(75 ꢃ 4.60 mm; 4 mm) at 25 ꢄC using a mobile phase A (water þ
155.56, 137.19, 130.80, 129.05, 119.01, 115.09, 32.44, 30.88. HRMS
(ESI): m/z [M þ H]þ calcd for C15H15N3O3S þ Hþ, 318.09069; found,
318.09066
1
0.1% TFA) and B (ACN þ 0.1% TFA) at a flow rate of 1 ml/min. H
2-Phenyl-1H-benzo[d]imidazole-6-sulfonamide (9)
spectra were recorded at 400 MHz on a Bruker Ascend 400 spec-
trometer while 13C NMR spectra were obtained by distortionless
enhancement by polarization transfer quaternary (DEPTQ) spec-
troscopy on the same spectrometer. Chemical shifts are reported
in d (ppm) relative to the internal reference tetramethylsilane
(TMS). Due to the existence of tautomers, some 1H and 13C NMR
signals could not be detected for some of the prepared benzimi-
dazoles so only the distinct signals are reported. Low resolution
mass spectra were recorded on a Finnigan LCQ DECA TermoQuest
mass spectrometer in electrospray positive and negative ionization
modes (ESI-MS). High resolution mass spectra were recorded on a
Bruker solariX MRMS in electrospray positive ionization modes
(ESI-FTMS). All tested compounds possessed a purity of at least
95% established by HPLC unless otherwise noted. Acids 27 and
28a were commercially available, acid 28b was obtained by previ-
ously reported procedure (see Supplementary Data).
Compound 9 was obtained as a light brown solid (49 mg, 75%) by
reaction of 26b (80 mg, 0.24 mmol) following the procedure
described for 6. H NMR (400 MHz, DMSO-d6) d 8.25–8.16 (m, 2H),
8.09 (d, J ¼ 1.8 Hz, 1H), 7.82 (d, J ¼ 8.7 Hz, 1H), 7.77 (dd, J ¼ 8.5,
1.8 Hz, 1H), 7.68–7.59 (m, 3H), 7.37 (s, 2H, exchangeable with D2O).
13C NMR (100 MHz, DMSO) d 153.36, 138.92, 131.25, 129.22,
128.00, 127.07, 120.64. HRMS (ESI): m/z [M þ H]þ calcd for
C13H11N3O2S þ Hþ, 274.06447; found, 274.06445.
1
Methyl 4-(6-sulfamoyl-1H-benzo[d]imidazol-2-yl)benzoate (10)
Compound 10 was obtained as a light brown solid (49 mg, 70%)
by reaction of 26c (82 mg, 0.212 mmol) following the procedure
described for 6. 1H NMR (400 MHz, DMSO-d6) 8.38–8.32 (m, 2H),
8.20–8.13 (m, 2H, 1H exchangeable with D2O), 8.00–7.85 (m, 1H),
7.78–7.68 (m, 1H), 7.33 (m, 2H, 1H exchangeable with D2O), 3.91
(s, 3H). 13C NMR (100 MHz, DMSO) d 165.71, 152.86, 152.43,
145.64, 142.87, 138.78, 138.31, 137.06, 134.23, 133.61, 130.84,
129.87, 126.96, 120.73, 119.74, 119.35, 117.09, 111.97, 109.84,
52.33. HRMS (ESI): m/z [M þ H]þ calcd for C15H13N3O4S þ Hþ,
332.06995; found, 332.06993.
2-(4-Hydroxyphenyl)-1H-benzo[d]imidazole-6-sulfonamide (6)
N-(tert-butyl)-2-(4-hydroxyphenyl)-1H-benzo[d]imidazole-6-sulfona-
mide 26a (174 mg, 0.504 mmol) was dissolved in 2 ml of a solution
DCM/TFA (1:1) and the mixture was stirred for 18 h. The solvent
was evaporated, and the resulting solid was crystallized with etha-
1
nol to give the title compound as a brown solid (110 mg, 75%). H
4-(6-Sulfamoyl-1H-benzo[d]imidazol-2-yl)benzoic acid (11)
NMR (400 MHz, DMSO-d6) d 10.12 (s, 1H, exchangeable with D2O),
8.03 (d, J ¼ 8.3 Hz, 2H), 7.99 (s, 1H), 7.72–7.64 (m, 2H), 7.28 (s, 2H,
exchangeable with D2O), 6.95 (d, J ¼ 8.3 Hz, 2H). 13C NMR
(100 MHz, DMSO) d 159.92, 154.19, 137.88, 128.67, 119.73, 115.85.
HRMS (ESI): m/z [M þ H]þ calcd for C13H11N3O3S þ Hþ, 290.05939;
found, 290.05938.
To a stirred solution of compound 10 (250 mg, 0.645 mmol) in
1.5 ml of THF was added a water solution (1.5 ml) of LiOH (62 mg,
2.58 mmol). The reaction mixture was stirred at room temperature
for 3 h and then concentrated under vacuum. The aqueous phase
was washed with CHCl3 then acidified with 3N HCl until a white
precipitate formed. After filtration, the title compound was
obtained as white solid (200 mg, 83%).1H NMR (400 MHz, DMSO-
d6) d 8.32 (d, J ¼ 8.2 Hz, 2H), 8.16–8.11 (m, 3H), 7.84–7.68 (m, 2H),
2-(4-Hydroxybenzyl)-1H-benzo[d]imidazole-6-sulfonamide (7)
Compound 7 was obtained as a white solid (47 mg, 75%) by reac- 7.32 (s, 2H, exchangeable with D2O). 13C NMR (100 MHz, DMSO) d
tion of 31 (74 mg, 0.206 mmol) following the procedure described 166.76, 133.24, 132.14, 129.99, 126.83. HRMS (ESI): m/z [M þ H]þ
for 6. 1H NMR (400 MHz, DMSO-d6) d 9.42 (s, 1H, exchangeable calcd for C14H11N3O4S þ Hþ, 318.05430; found, 318.05429.
with D2O), 8.04 (s, 1H), 7.79–7.73 (m, 2H), 7.39 (s, 2H, exchange-
able with D2O), 7.16 (d, J ¼ 8.0 Hz, 2H), 6.75 (d, J ¼ 7.8 Hz, 2H), 4.26
(s, 2H). 13C NMR (100 MHz, DMSO) d 156.71, 156.58, 139.31,
2-(3,4-Dihydroxyphenyl)-1H-benzo[d]imidazole-6-sulfonamide (12)
Compound 12 was obtained as a light brown solid (105 mg, 73%)
by reaction of 26d (170 mg, 0.470 mmol) following the procedure
described for 6. 1H NMR (400 MHz, DMSO-d6) d 9.54 (s, 1H,
exchangeable with D2O), 9.31 (s, 1H, exchangeable with D2O),
8.02, 7.90 (2 s, 1H), 7.73–7.55 (m, 3H), 7.48 (dd, J ¼ 8.4, 1.7 Hz, 1H),
7.31–7.17 (m, 2H, exchangeable with D2O), 6.89 (d, J ¼ 8.5 Hz, 1H).
13C NMR (100 MHz, DMSO) d 148.09, 145.63, 143.10, 137.50,
120.73, 119.53, 119.16, 118.58, 118.18, 116.13, 115.80, 114.26,
110.98, 109.09. HRMS (ESI): m/z [M þ H]þ calcd for
C13H11N3O4S þ Hþ, 306.05430; found, 306.05431.
130.02, 125.27, 120.94, 115.54, 114.50, 112.54, 32.95. HRMS (ESI):
m/z [M þ H]þ calcd for C14H13N3O3S þ Hþ, 304.07504; found,
304.07503.
2-(4-Hydroxyphenethyl)-1H-benzo[d]imidazole-6-sulfonamide (8)
Compound 30a (245 mg, 0.626 mmol) was dissolved in 80 ml of
toluene, p-toluensulfonic acid (59 mg, 0.313 mmol) was added and
the resulting mixture was heated at reflux for 6 h. Solvent was
evaporated and the crude residue was taken up with a saturated
solution of NaHCO3 (30 ml). The aqueous phase was extracted
with EtOAc (3 ꢃ 20 ml) and the collected organic phases were
washed with saturated solution of NaHCO3 (3 ꢃ 20 ml), brine
(20 ml), anhydrified over Na2SO4, filtered and concentrated under
Methyl
2-hydroxy-5-(6-sulfamoyl-1H-benzo[d]imidazol-2-yl)ben-
zoate (13)
reduced pressure. The title compound was obtained after crystal- Compound 13 was obtained as a light brown solid (38 mg, 75%)
lization with ethanol as a light brown solid (130 mg, 65%). 1H by reaction of 26e (60 mg, 0.149 mmol) following the procedure
NMR (400 MHz, DMSO-d6) d 9.15 (s, 1H, exchangeable with D2O), described for 6. 1H NMR (400 MHz, DMSO-d6) d 10.94 (s, 1H,

1700
C. MILITE ET AL.
exchangeable with D2O), 8.65 (d, J ¼ 2.3 Hz, 1H), 8.32 (dd, J ¼ 8.8, 2-Hydroxy-5-(2-(6-sulfamoyl-1H-benzo[d]imidazol-2-yl)ethyl)ben-
2.2 Hz, 1H), 8.04 (s, 1H), 7.80–7.70 (m, 2H), 7.35 (s, 2H, exchange-
able with D2O), 7.23 (dd, J ¼ 8.8, 2.0 Hz, 1H), 3.97 (s, 3H). 13C NMR
(100 MHz, DMSO) d 168.12, 161.55, 152.68, 138.60, 133.57, 129.30,
120.36, 118.50, 114.47, 52.64. HRMS (ESI): m/z [M þ H]þ calcd for
C15H13N3O5S þ Hþ, 348.06487; found, 348.06486.
zoic acid (18)
Compound 18 was obtained as a white solid (135 mg, 93%) by
reaction of 17 (150 mg, 0.400 mmol) following the procedure
described for 11. 1H NMR (400 MHz, DMSO-d6) d 8.14 (s, 1H),
7.91–7.89 (m, 2H), 7.70 (d, J ¼ 2.4 Hz, 1H), 7.51 (s, 2H, exchange-
able with D2O), 7.47–7.37 (m, 1H), 6.89 (d, J ¼ 8.5 Hz, 1H), 3.39 (t,
J ¼ 7.8 Hz, 2H), 3.18 (t, J ¼ 7.8 Hz, 2H). 13C NMR (100 MHz, DMSO) d
171.64, 159.74, 156.30, 140.72, 135.63, 129.98, 129.65, 122.28,
117.32, 114.47, 112.82, 112.00, 31.19, 28.83.HRMS (ESI): m/z
[M þ H]þ calcd for C16H15N3O5S þ Hþ, 362.08052; found,
362.08052.
2-hydroxy-5-(6-sulfamoyl-1H-benzo[d]imidazol-2-yl)benzoic
acid (14)
Compound 14 was obtained as white solid (120 mg, 83%) by reac-
tion of 13 (150 mg, 0.43 mmol) following the procedure described
1
for 11. H NMR (400 MHz, DMSO-d6) d 8.68 (d, J ¼ 2.4 Hz, 1H), 8.32
(dd, J ¼ 8.7, 2.5 Hz, 1H), 8.02 (s, 1H), 7.75–7.67 (m, 2H), 7.30 (s, 2H,
exchangeable with D2O), 7.17 (dd, J ¼ 8.8, 2.2 Hz, 1H). 13C NMR
(100 MHz, DMSO) d 171.28, 163.06, 153.18, 138.07, 133.57, 129.08,
119.88, 118.11, 114.04. HRMS (ESI): m/z [M þ H]þ calcd for
C14H11N3O5S þ Hþ, 334.04922; found, 334,04922.
5-(6-Carbamoyl-1H-benzo[d]imidazol-2-yl)-2-hydroxybenzoic
acid (19)
Compound 35 (150 mg, 0.408 mmol) was dissolved in 2 ml of a
solution DCM/TFA (1:1) and the mixture was stirred for 24 h. The
solvent was evaporated, and the resulting solid was taken up with
2 ml of THF. To the resulting mixture, a water solution (2 ml) of
LiOH (62 mg, 2.58 mmol) was added, and the reaction mixture was
stirred at room temperature for 4 h. The reaction was concen-
trated under vacuum, the aqueous phase was washed with CHCl3
then acidified with 3N HCl until a white precipitate formed that
was recovered by filtration. Compound 19 was obtained as white
solid (110 mg, 91%) after recrystallization from ethanol 1H NMR
(400 MHz, DMSO-d6) d 8.72 (d, J ¼ 2.4 Hz, 1H), 8.36–8.26 (m, 1H),
8.17 (s, 1H), 8.09 (s, 1H, exchangeable with D2O), 7.88 (d,
J ¼ 8.4 Hz, 1H), 7.71–7.65 (m, 1H), 7.38 (s, 1H, exchangeable with
D2O), 7.19 (d, J ¼ 8.7 Hz, 1H). 13C NMR (100 MHz, DMSO) d 171.54,
168.31, 164.41, 152.09, 134.20, 130.25, 130.08, 123.63, 118.84,
115.01. HRMS (ESI): m/z [M þ H]þ calcd for C15H11N3O4 þ Hþ,
298.08223; found, 298.08230.
Methyl 5-(6-(N-ethylsulfamoyl)-1H-benzo[d]imidazol-2-yl)-2-hydrox-
ybenzoate (15)
Compound 15 was obtained as a light brown solid (235 mg, 71%)
by reaction of 20b (188 mg, 0.88 mmol) and methyl 5-formylsalicy-
late (160 mg, 0.88 mmol) following the procedure described for 6.
1H NMR (400 MHz, DMSO-d6) d 10.85 (s, 1H, exchangeable with
D2O), 8.65 (d, J ¼ 2.4 Hz, 1H), 8.32 (dd, J ¼ 8.7, 2.4 Hz, 1H),
7.99–7.96 (m, 1H), 7.79–7.68 (m, 1H), 7.68–7.59 (m, 1H), 7.47 (t,
J ¼ 5.7 Hz, 1H, exchangeable with D2O), 7.20 (d, J ¼ 8.7 Hz, 1H),
3.97 (s, 3H), 2.83 – 2.72 (m, 2H), 0.96 (t, J ¼ 7.2 Hz, 3H). 13C NMR
(100 MHz, DMSO) d 168.31, 161.28, 153.22, 133.98, 133.47, 128.91,
120.80, 120.49, 118.38, 114.19, 52.62, 37.55, 14.65. HRMS (ESI): m/z
[M þ H]þ calcd for C17H17N3O5S þ Hþ, 376.09617; found, 376.09620.
5-(6-(N-ethylsulfamoyl)-1H-benzo[d]imidazol-2-yl)-2-hydroxyben-
zoic acid (16)
3,4-Diamino-N-(tert-butyl)benzenesulfonamide (20a)
Compound 16 was obtained as a white solid (128 mg, 89%) by
reaction of 15 (150 mg, 0.40 mmol) following the procedure
described for 11. 1H NMR (400 MHz, DMSO-d6) 8.71 (d, J ¼ 2.3 Hz,
1H), 8.36 (dd, J ¼ 8.8, 2.3 Hz, 1H), 8.01 (s, 1H), 7.79 (d, J ¼ 8.5 Hz,
1H), 7.69 (dd, J ¼ 8.5, 1.8 Hz, 1H), 7.55 (t, J ¼ 5.9 Hz, 1H, exchange-
able with D2O), 7.21 (d, J ¼ 8.8 Hz, 1H), 2.85–2.72 (m, 2H), 0.97 (t,
J ¼ 7.3 Hz, 3H). 13C NMR (100 MHz, DMSO) d 171.17, 163.22, 152.94,
134.66, 133.87, 129.51, 121.09, 118.27, 114.01, 37.56, 14.66. HRMS
(ESI): m/z [M þ H]þ calcd for C16H15N3O5S þ Hþ, 362.08052; found,
362.08061.
To a stirred suspension of 24a (1.65 g, 6.04 mmol) in 250 ml of
MeOH, ammonium formate (7.61 g, 120.74 mmol) and palladium
on carbon 10% wt. (160 mg) were added. The resulting mixture
was heated at reflux for 4 h. After cooling, the mixture was fil-
tered, and the solvent evaporated under reduced pressure. The
crude material was taken up with 100 ml of water and extracted
with EtOAc (3 ꢃ 60 ml). The combined organic phases were
washed with brine, dried over Na2SO4, filtered and evaporated.
The product, obtained as a light brown solid (1.30 g, 88%), was
used for the next step without further purification. 1H NMR
(400 MHz, DMSO-d6) d 6.96 (d, J ¼ 2.1 Hz, 1H), 6.90–6.84 (m, 2H, 1H
exchangeable with D2O), 6.53 (d, J ¼ 8.2 Hz, 1H), 5.13 (brs, 2H,
exchangeable with D2O), 4.79 (brs, 2H, exchangeable with D2O),
1.08 (s, 9H). ESI m/z: 244 [M þ H]þ.
Methyl
2-hydroxy-5-(2-(6-sulfamoyl-1H-benzo[d]imidazol-2-yl)e-
thyl)benzoate (17)
Compound 17 was obtained as a white solid (285 mg, 60%) by
reaction of 30b (570 mg, 1.27 mmol) following the procedure
described for 8. 1H NMR (400 MHz, DMSO-d6) d 10.32 (s, 1H,
exchangeable with D2O) , 7.98, 7.87 (2 s, 1H), 7.68–7.54 (m, 3H), 3,4-Diamino-N-ethylbenzenesulfonamide (20b)
7.39 (dd, J ¼ 8.5, 2.4 Hz, 1H), 7.25–7.19 (m, 2H, exchangeable with Compound 20b was obtained as a light brown solid (910 mg,
D2O), 6.89 (d, J ¼ 8.4 Hz, 1H), 3.85 (s, 3H), 3.15–3.04 (m, 4H). 13C 94%) by reaction of 24b (1.10 g, 4.48 mmol) following the proced-
NMR (100 MHz, DMSO) d 169.13, 158.41, 157.48, 156.87, 145.32, ure described for 20a. 1H NMR (400 MHz, DMSO-d6) d 6.94–6.88
142.42, 137.19, 136.26, 135.75, 131.61, 129.27, 119.29, 118.73, (m, 2H, 1H exchangeable with D2O), 6.81 (dd, J ¼ 8.0, 2.1 Hz, 1H),
118.11, 117.36, 116.10, 112.64, 110.93, 109.07, 52.34, 32.08, 30.52. 6.53 (d, J ¼ 8.0 Hz, 1H), 5.18 (brs, 2H, exchangeable with D2O), 4.82
HRMS (ESI): m/z [M þ H]þ calcd for C17H17N3O5S þ Hþ, 376.09617; (brs, 2H, exchangeable with D2O), 2.75–2.62 (m, 2H), 0.94 (t,
found, 376.09613.
J ¼ 7.2 Hz, 3H). ESI m/z: 216 [M þ H]þ.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
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Ethyl 2-((2-nitrophenyl)amino)-2-oxoacetate (22)
To solution of 2-nitroaniline (2.00 g, 14.48 mmol) in Et2O
(s, 1H, exchangeable with D2O), 8.03 (d, J ¼ 8.5 Hz, 2H), 8.01–7.96
(m, 1H), 7.69–7.61 (m, 2H), 7.43 (s, 1H, exchangeable with D2O),
6.94 (d, J ¼ 8.5 Hz, 2H), 1.09 (s, 9H).ESI m/z: 346 [M þ H]þ.
a
(100 ml), ethyl chlorooxoacetate (2.17 g, 1.78 ml, 15.93 mmol) was
added portionwise with continuous stirring. Once the addition
was complete, the resulting yellow suspension was stirred for 18 h
at room temperature and then concentrated under vacuo. The
crude residue was dissolved in EtOAc (100 ml), washed with satu-
rated NaHCO3 (3 ꢃ 30 ml) and with brine (30 ml). The organic
N-(tert-butyl)-2-phenyl-1H-benzo[d]imidazole-6-sulfonamide (26b)
Compound 26b was obtained as a light brown solid (204 mg,
65%) by reaction of 20a (233 mg, 0.954 mmol) and benzaldehyde
1
phase was dried over anhydrous Na2SO4, and evaporated to dry- (97 mL, 0.954 mmol) following the procedure described for 26a. H
1
ness, giving the desired product as a yellow solid (3.38 g, 98%). H
NMR (400 MHz, DMSO-d6) d 11.38 (s, 1H), 8.17–8.05 (m, 2H),
7.85–7.76 (m, 1H), 7.52–7.41 (m, 1H), 4.34 (q, J ¼ 7.0 Hz, 2H), 1.33
(t, J ¼ 7.0 Hz, 3H). ESI m/z: 239 [M þ H]þ.
NMR (400 MHz, DMSO-d6) d 8.23–8.15 (m, 2H), 8.11–8.04 (m, 1H),
7.85–7.72 (m, 2H), 7.68–7.58 (m, 3H), 7.54 (s, 1H, exchangeable
with D2O), 1.09 (s, 9H).ESI m/z: 330 [M þ H]þ.
Methyl 4-(6-(N-(tert-butyl)sulfamoyl)-1H-benzo[d]imidazol-2-yl)ben-
zoate (26c)
4-Amino-3-nitrobenzenesulfonyl chloride (23)
A solution of ethyl 2-(2-nitrophenylamino)-2-oxoacetate 22 (2.00 g,
8.40 mmol) in 4.5 ml of chlorosulfonic acid was heated at 80 ꢄC for
3 h. The red mixture was poured slowly into ice ꢁ water (150 ml)
and stirred for 30 min. The product was extracted from the aque-
ous solution using Et2O (3 ꢃ 30 ml). The combined organic phases
were washed with brine (10 ml), dried (Na2SO4), filtered, and con-
centrated in vacuo to give the title compound as a brown solid
which was immediately used for the next reaction without purifi-
Compound 26c was obtained as a light brown solid (190 mg,
83%) by reaction of 20a (144 mg, 0.590 mmol) and methyl 4-for-
mylbenzoate (97 mg, 0.590 mmol) following the procedure
described for 26a. 1H NMR (400 MHz, DMSO-d6) d 8.35 (d,
J ¼ 8.0 Hz, 2H), 8.17 (d, J ¼ 8.0 Hz, 2H), 8.09 (s, 1H), 7.83–7.70 (m,
2H), 7.51 (s, 1H, exchangeable with D2O), 3.91 (s, 3H), 1.10 (s, 9H).
ESI m/z: 388 [M þ H]þ.
1
cation. H NMR (400 MHz, DMSO-d6) d 8.16 (d, J ¼ 2.0 Hz, 1H), 7.56
(dd, J ¼ 8.8, 2.0 Hz, 1H), 6.96 (d, J ¼ 8.8 Hz, 1H).
N-(tert-butyl)-2-(3,4-dihydroxyphenyl)-1H-benzo[d]imidazole-6-sul-
fonamide (26d)
Compound 26d was obtained as a light brown solid (290 mg,
65%) by reaction of 20a (300 mg, 1.23 mmol) and 3,4-dihydroxy-
benzaldehyde (170 mg, 1.23 mmol) following the procedure
described for 26a. 1H NMR (400 MHz, DMSO-d6) d 9.42 (brs, 2H,
exchangeable with D2O), 8.02, 7.90 (2 s, 1H), 7.73–7.58 (m, 3H),
7.49 (dd, J ¼ 8.2, 2.1 Hz, 1H), 7.42 (s, 1H, exchangeable with D2O),
6.90 (d, J ¼ 8.3 Hz, 1H), 1.09 (s, 9H).ESI m/z: 362 [M þ H]þ.
4-Amino-N-(tert-butyl)-3-nitrobenzenesulfonamide (24a)
To a stirred solution at 0 ꢄC of crude 23 (1.04 g, 4.39 mmol) in dry
THF (25 ml) was added dropwise, under nitrogen atmosphere, tert-
butylamine (1.85 ml, 17.56 mmol). The reaction was allowed to
reach room temperature and was stirred for 18 h. The solvent was
removed at reduced pressure and the residue was taken up with
50 ml of water and extracted with EtOAc (3 ꢃ 20 ml). The com-
bined organic phases were washed with brine, dried over Na2SO4,
filtered and evaporated under reduced pressure. Purification by
silica gel chromatography (DCM/MeOH) yield pure 24a (0.96 g,
80%) as a light yellow solid. 1H NMR (400 MHz, DMSO-d6) d 8.38
(d, J ¼ 2.2 Hz, 1H), 7.95 (s, 2H, exchangeable with D2O), 7.70 (dd,
Methyl 5-(6-(N-(tert-butyl)sulfamoyl)-1H-benzo[d]imidazol-2-yl)-2-
hydroxybenzoate (26e)
Compound 26e was obtained as a light brown solid (222 mg,
J ¼ 9.0, 2.2 Hz, 1H), 7.44 (s, 1H, exchangeable with D2O), 7.11 (d, 71%) by reaction of 20a (188 mg, 0.773 mmol) and methyl 5-for-
J ¼ 9.0 Hz, 1H), 1.10 (s, 9H). ESI m/z: 274 [M þ H]þ.
mylsalicylate (139 mg, 0.773 mmol) following the procedure
described for 26a. 1H NMR (400 MHz, DMSO-d6) d 8.65 (d,
J ¼ 2.3 Hz, 1H), 8.32 (dd, J ¼ 8.7, 2.3 Hz, 1H), 8.01 (s, 1H), 7.76–7.64
4-Amino-N-ethyl-3-nitrobenzenesulfonamide (24b)
Compound 24b was obtained as a yellow solid (840 mg, 78%) by
reaction of 23 (1.04 g, 4.39 mmol) with a 2 M THF solution of ethyl-
(m, 2H), 7.46 (s, 1H, exchangeable with D2O), 7.19 (d, J ¼ 8.7 Hz,
1H), 3.97 (s, 3H), 1.09 (s, 9H). ESI m/z: 404 [M þ H]þ.
amine (8.78 ml, 17.56 mmol) following the procedure described for
1
24a. H NMR (400 MHz, DMSO-d6) d 8.36 (d, J ¼ 1.6 Hz, 1H), 8.01 (s,
N-(2-amino-4-(N-(tert-butyl)sulfamoyl)phenyl)-2-(4-hydroxypheny-
2H, exchangeable with D2O), 7.69 (dd, J ¼ 9.0, 1.6 Hz, 1H),
l)acetamide and N-(2-amino-5-(N-(tert-butyl)sulfamoyl)phenyl)-2-
7.54–7.44 (m, 1H, exchangeable with D2O), 7.15 (d, J ¼ 9.0 Hz, 1H),
(4-hydroxyphenyl)acetamide (29)
2.82 – 2.72 (m, 2H), 0.99 (t, J ¼ 7.2 Hz, 3H). ESI m/z: 246 [M þ H]þ.
To a stirred solution of 20a (354 mg, 1.45 mmol) and 4-hydroxy-
phenylacetic acid 27 (200 mg, 1.31 mmol) in dry DMF (26 ml) were
added, EDC hydrochloride (427 mg, 2.23 mmol), HOBt (341 mg,
2.23 mmol) and 4-methylmorpholine (4.84 ml, 4.45 mmol). The
reaction was stirred at room temperature for 18 h. Water (80 ml)
was added the resulting mixture was extracted with EtOAc
N-(tert-butyl)-2-(4-hydroxyphenyl)-1H-benzo[d]imidazole-6-sulfona-
mide (26a)
To a stirred solution of 20a (150 mg, 0.62 mmol) in dry DMF (7 ml),
4-hydroxybenzaldehyde (75 mg, 0.61 mmol) and Na2S2O5 (0.165 g,
(3 ꢃ 40 ml). The combined organic phases were washed with brine
0.793 mmol) were added. The resulting mixture was stirred at
80 ꢄC for 18 h. After cooling at room temperature, water was
added. The brown precipitate formed was recovered by filtration
and was washed several times with water and 1N HCl. After
recrystallization from EtOH, compound 26a was obtained as light
(3 ꢃ 50 ml), dried over Na2SO4, filtered and evaporated under
reduced pressure. Purification by silica gel chromatography (DCM/
MeOH) yield an isomeric mixture as a white solid (0.360 g, 73%)
that was used for the next step without separation. ESI m/z:
brown solid (160 mg, 72%). 1H NMR (400 MHz, DMSO-d6) d 10.07 378 [M þ H]þ.

1702
C. MILITE ET AL.
N-(2-amino-4-(N-(tert-butyl)sulfamoyl)phenyl)-3-(4-hydroxyphenyl)-
Methyl
5-(6-(tert-butylcarbamoyl)-1H-benzo[d]imidazol-2-yl)-2-
propanamide and N-(2-amino-5-(N-(tert-butyl)sulfamoyl)phenyl)-3- hydroxybenzoate (35)
Compound 35 was obtained as a light brown solid (304 mg, 72%)
(4-hydroxyphenyl)propanamide (30a)
by reaction of 34 (240 mg, 1.15 mmol) and 25e (208 mg,
1.15 mmol) following the procedure described for 6. 1H NMR
(400 MHz, DMSO-d6) d 11.04 (s, 1H, exchangeable with D2O), 8.66
(d, J ¼ 2.2 Hz, 1H), 8.30 (dd, J ¼ 8.8, 2.4 Hz, 1H), 8.09 (s, 1H),
7.90–7.78 (m, 2H, 1H exchangeable with D2O), 7.68 (d, J ¼ 8.4 Hz,
1H), 7.26 (d, J ¼ 8.7 Hz, 1H), 3.97 (s, 3H), 1.42 (s, 9H). ESI m/z:
Compounds 30a was obtained as a light yellow solid (300 mg,
64%) by reaction of 20a (323 mg, 1.33 mmol) with 3-(4-hydroxy-
phenyl)propanoic acid 28a (200 mg, 1.2 mmol) following the pro-
cedure described for 29. ESI m/z: 392 [M þ H]þ.
Methyl 5-(3-((2-amino-4-(N-(tert-butyl)sulfamoyl)phenyl)amino)-3- 368 [M þ H]þ.
oxopropyl)-2-hydroxybenzoate and methyl 5-(3-((2-amino-5-(N-
(tert-butyl)sulfamoyl)phenyl)amino)-3-oxopropyl)-2-hydroxyben-
Enzyme activity assays
zoate (30b)
An Applied Photophysics stopped-flow instrument has been used
for assaying the CA-catalysed CO2 hydration activity18. Phenol red
(at a concentration of 0.2 mM) has been used as indicator, working
at the absorbance maximum of 557 nm, with 20 mM Hepes (pH
7.5) as buffer, and 20 mM Na2SO4 (for maintaining constant the
ionic strength), following the initial rates of the CA-catalysed CO2
hydration reaction for a period of 10–100 s. CO2 concentrations
ranged from 1.7 to 17 mM for the determining inhibition con-
stants. For each inhibitor, at least six traces of the initial 5–10% of
the reaction have been used for measuring the initial velocity.
Uncatalyzed rates were determined in the same manner and sub-
tracted from the total observed rates. Stock solutions of inhibitor
(0.1 mM) were prepared in buffer with a maximum 3% DMSO, and
dilutions up to 0.01 nM were done with the assay buffer. Inhibitor
and enzyme solutions were preincubated for 15 min at room tem-
perature prior to assay, in order to allow for the formation of the
E–I complex. The inhibition constants were obtained by nonlinear
least-squares methods using PRISM 3 and the Cheng-Prusoff equa-
tion, as reported earlier, and represent the mean from at least
three different determinations19–26. All CA isoforms were recom-
Compounds 30b was obtained as a light yellow solid (90 mg,
90%) by reaction of 20a (60 mg, 0.245 mmol) with 3-(4-hydroxy-3-
(methoxycarbonyl)phenyl)propanoic acid 28b (50 mg, 0.223 mmol)
following the procedure described for 29. ESI m/z: 450 [M þ H]þ.
N-(tert-butyl)-2-(4-hydroxybenzyl)-1H-benzo[d]imidazole-6-sulfona-
mide (31)
In a 10 ml CEM pressure vessel equipped with a stirrer bar, com-
pounds 29 (50 mg, 0.132 mmol) were dissolved in 1.3 ml of acetic
acid. The microwave vial was sealed and heated in a CEM
Discover microwave synthesizer to 80 ꢄC for 30 min. After cooling
to room temperature, the reaction mixture was taken up with
solution of NaHCO3 (60 ml) and the aqueous phase was extracted
with EtOAc (3 ꢃ 20 ml). The collected organic phases were washed
with water (3 ꢃ 20 ml), NaHCO3 solution (3 ꢃ 20 ml), brine (20 ml),
dried over Na2SO4 and filtered. The solvent was removed under
reduced pressure and the resulting crude material was purified by
silica gel chromatography (DCM/EtOAc) to give compound 31 a
light brown solid (35 mg, 74%). 1H NMR (400 MHz, DMSO-d6)
d 9.31 (s, 1H, exchangeable with D2O), 7.93 (s, 1H), 7.64–7.54 (m, 2H),
7.39 (s, 1H, exchangeable with D2O), 7.14 (d, J¼ 8.0 Hz, 2H), 6.71
(d, J¼ 8.0 Hz, 2H), 4.09 (s, 2H), 1.06 (s, 9H). ESI m/z: 360 [Mþ H]þ.
binant ones obtained in-house as reported earlier27–29
.
Molecular modeling methods
The latest version of the AD4 docking software (version 4.2)30
together with its GUI AutoDockTools (ADT) and the
AutoDock4(Zn) force field31, were employed. The hCA IX X-ray
structure used for the experiment had the PDB code 5FL432. The
protein structure was prepared for the docking using the Protein
Preparation Wizard of the Maestro suite33 that adds bond orders,
adds hydrogen atoms, deletes water molecules and produces the
appropriate protonation states. The co-crystal ligand of 5FL4 was
separated from the cognate protein. The 2 D Sketcher tool of
Maestro was used to build compounds 13, 14 and 17. For the
three ligands, the protonation and tautomeric state, as well as
N-(tert-butyl)-3,4-dinitrobenzamide (33)
A solution of 3,4-dinitrobenzoic acid 32 (1.00 g, 4.71 mmol) in thi-
onyl chloride (15 ml) was refluxed for 2 h under a nitrogen atmos-
phere. After the solution was cooled at room temperature, the
excess thionyl chloride was removed at reduced pressure and the
crude material dried under vacuum. To the residue, dissolved in
dry THF (20 ml) and cooled to 0 ꢄC, was added dropwise a mixture
of tert-butylamine (525 mL, 5.00 mmol) and triethylamine (695 mL,
5.00 mmol) in dry THF (5 ml). The mixture was stirred at room tem-
perature for 18 h, filtered, and evaporated. The crude residue was
dissolved in DCM (20 ml), washed with 1N HCl, saturated NaHCO3, their geometry, were optimized through LigPrep, part of the same
suite. Through Maestro, the X-ray structures of hCA I (PDB 6F3B)34,
and water, dried, and concentrated in vacuum. Recrystallization
hCA II (PDB 3K34)35, and hCA XII (PDB 5MSA)36, were downloaded
and superimposed on the structure of hCA IX. The ligands were
translated in the AD4 specific file format (PDBQT) using the
from EtOH yielded compound 33 (1.05 g, 84%) as a light yellow
solid. 1H NMR (400 MHz, DMSO-d6) d 8.57 (s, 1H), 8.35–8.29 (m,
2H), 1.40 (s, 9H). ESI m/z: 268 [M þ H]þ.
python scripts prepare_ligand4.py and prepare_receptor4.py, part
of ADT, applying the standard settings. Following the
AutoDock4(Zn) force field protocol37, to add the tetrahedral zinc
3,4-Diamino-N-(tert-butyl)benzamide (34)
Compound 34 was obtained as a light brown solid (729 mg, 94%)
by reaction of 33 (1.00 g, 3.74 mmol) following the procedure
described for 20a. 1H NMR (400 MHz, DMSO-d6) d 7.05 (s, 1H,
exchangeable with D2O), 6.98 (d, J ¼ 2.0 Hz, 1H), 6.90 (dd, J ¼ 8.1,
2.1 Hz, 1H), 6.45 (d, J ¼ 8.0 Hz, 1H), 4.86 (s, 2H, exchangeable with
pseudo atoms to the receptor PDBQT the script zinc_pseudo.py,
part of the material provided with the force field, was employed.
The docking area was centered on the active site. The zinc-specific
non bonded pairwise potentials were included in the creation of
the grid parameter file. A set of grids of 60 Å ꢃ 40 Å ꢃ 50 Å with
D2O), 4.48 (s, 2H, exchangeable with D2O), 1.33 (s, 9H). ESI m/z: 0.375 Å spacing was calculated considering the docking area for
208 [M þ H]þ.
all the ligands atom types employing AutoGrid4. For every ligand,

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1703
200 independent docking simulations were achieved. Each dock- furnished the amides 29, 30a and 30b as regioisomeric mixtures,
ing calculation comprised 20 million energy evaluations employ- which were purified without separation of regioisomers.
ing the Lamarckian genetic algorithm local search (GALS) method.
This latter assesses a population of viable docking solutions and
propagates the best individuals from each generation into the fol-
lowing generation of feasible solutions. A low-frequency local
search according to the method of Solis and Wets was applied to
every docking attempt to guarantee that the final solution repre-
sented a local minimum. All dockings were performed with a
population size of 250, and 300 iterations of Solis and Wets local
search were applied with a probability of 0.06. A rate of mutation
of 0.02 and a crossover rate of 0.8 were used to produce new
docking attempts for following generations, and the best individ-
ual from each generation was propagated over the following gen-
eration. The docking results from every (200) independent
docking calculation were clustered based on the of root-mean-
square deviation (rmsd) (solutions differing by less than 2.0 Å)
between the Cartesian coordinates of the atoms and were ranked
on the basis of free energy of binding (DGAD4).
Benzimidazoles derivatives
8 and 17 were straightforwardly
obtained by p-toluenesulfonic acid-mediated cyclization and
deprotection of the corresponding 2-amido anilines 30a and 30b
in refluxing toluene. The carboxylic acid 18 was obtained by
deprotecting the methyl ester 17 with lithium hydroxide. Our
attempts to cyclize compound 29 using the same reaction condi-
tions were not successful. On the other hand, benzimidazole 31
was obtained in good yield (74%) by using acetic acid under
microwave irradiation. Finally, deprotection with trifluoracetic acid
at room temperature furnished the primary sulfonamide 7.
N-ethyl-2-aryl-1H-benzo[d]imidazole-6-sulfonamides 15 and 16
were obtained starting from 3,4-diamino-N-ethylbenzenesulfona-
mide 20b and aldehyde 25e following the same synthetic strategy
used for the preparation of the primary sulfonamide analogs 13
and 14 (Scheme 3).
Finally, 2–(4-hydroxy-3-carboxy)-phenyl-1H-benzo[d]imidazole-6-
carboxamide 19 was straightforwardly synthesized as described in
Scheme 4. After activation of the commercially available 3,4-dini-
trobenzoic acid 32 with thionyl chloride, coupling with tert-butyl
amine yielded 3,4-dinitrobenzamide 33 which was reduced by
catalytic hydrogenation with ammonium formate and palladium
catalyst to the key intermediate 34. Condensation with aldehyde
25e in the presence of NaHSO3 gave the benzimidazole derivative
35. Amide deprotection with trifluoracetic acid and hydrolysis of
the methyl ester with LiOH gave the desired compound 19.
Results and discussion
Chemistry
The primary (6–14 and 17–18) and the N-ethyl-2-substituted-1H-
benzo[d]imidazole-6-sulfonamides (15 and 16) were synthesized
starting from 3,4-diaminobenzenesulfonamides 20a and 20b,
respectively, obtained following the procedure outlined in
Scheme 1.
The amino group of 2-nitroaniline 21 was protected by acyl-
ation with ethyl chlorooxoacetate in diethyl ether. Reaction of the
resulting ethyl 2-(2-nitrophenylamino)-2-oxoacetate 22 with chlor-
osulfonic acid at 80 ꢄC, followed by aqueous workup, yielded the
unprotected sulfonyl chloride derivative 23, which reacted with
tert-butyl- or ethyl-amine to afford the N-substituted-4-amino-3-
nitrobenzenesulfonamides 24a and 24b, respectively. Catalytic
hydrogenation with ammonium formate and palladium catalyst
converted the nitro derivatives into the corresponding amino
derivatives 20a and 20b.
The synthetic route to 2-aryl-1H-benzo[d]imidazole-6-sulfona-
mides 6–14 is outlined in Scheme 2. Condensation of 3,4-dia-
mino-N-(tert-butyl)benzenesulfonamide 20a with aldehydes 25a-e
in the presence of NaHSO3 in dry DMF at 80 ꢄC gave the 2-aryl-N-
(tert-butyl)-1H-benzo[d]imidazole-6-sulfonamides 26a-e in good
yields (65–83%)38. Deprotection with trifluoracetic acid at room
temperature furnished the primary sulfonamides 6, 9, 10, 12 and
13. Finally, the carboxylic acid derivatives 11 and 14 were
obtained by deprotecting the methyl esters 10 and 13, respect-
ively, with lithium hydroxide.
CA inhibition assays and structure-activity relationship (SAR)
considerations
Table 1 lists the enzyme inhibitory activities of the newly synthe-
sized compounds 6–19 against the human (h) CA I, II, IX and XII
isoforms, assessed by a stopped-flow CO2 hydrase assay [19]. AAZ
1 was used as the standard drug in the assay18. Selectivity ratios
(SR) for inhibiting the tumor-associated transmembrane isoforms
(hCA IX and XII) over the physiologically dominant cytosolic one
(hCA II) are also reported for the most active compounds.
First, as putative leads for the development of selective CAIs,
we synthesized compound 6 and its homologs with a methyl or
ethyl linker between the benzimidazole and the phenol moieties
(compounds 7 and 8 in Table 1). They all resulted in medium-
potency inhibition of the slow cytosolic isoform hCA I, with Ki val-
ues ranging from 213.6 nM to 442.1 nM. The 40-hydroxybenzyl
derivative 7 showed good inhibition activity for hCA II, IX, and XII
but was not selective (Ki 91.9 nM for hCA II, 73.9 nM for hCA IX,
63.8 nM for hCA XII). On the other hand, the 40-hydroxyphenyl (6)
and 40-hydroxyphenylethyl (8) analogs were effective inhibitors of
hCA IX and XII, respectively, with Kis in the low nanomolar range
(6, Ki 17.4 nM for hCA IX, 44.1 nM for hCA XII; 8, Ki 14.4 nM for
hCA IX, 9.8 nM for hCA XII) basically comparable to those of the
reference 1 (Ki 25 nM for hCA IX, 5.7 nM for hCA XII), and interest-
Coupling reaction of benzenesulfonamide 20a with 2-(4-
hydroxyphenyl)acetic acid 27 or 3-arylpropanoic acids 28a and
28b, in the presence of the peptide coupling reagents hydroxy-
benzotriazole (HOBt) and 1-ethyl-3–(3-dimethylaminopropyl)carbo-
diimide hydrochloride (EDC) and N-methylmorpholine in dry DMF, ing selectivity ratio (SR) vs hCA II.
Scheme 1. (a) Ethyl chlorooxoacetate, Et2O, r.t. 18 h (98%); (b) ClSO3H, 80 ꢄC, 3 h (77%); c) tert-butylamine or 2M THF solution of ethylamine, THF, 0 ꢄC to r.t., 18 h
(80%); (d) ammonium formate, Pd/C 10%, MeOH, reflux, 4 h (88–94%).

1704
C. MILITE ET AL.
Scheme 2. (a) NaHSO3, dry DMF, 80 ꢄC, 18h (65%–83%); (b) DCM/TFA (1:1), r.t., 18–24 h (70–75%); (c) LiOH, THF/H2O (1:1) r.t, 3 h (83–93%); (d) EDC hydrochloride,
HOBt, NMM, dry DMF, r.t., 18h (64–90%); (e) p-toluenesulfonic acid, toluene, reflux, 6 h (60–65%); (f) MW, AcOH, 80 ꢄC, 30 min (74%).
Scheme 3. (a) 25e, NaHSO3, dry DMF, 80ꢄC, 18 h (71%); (b) LiOH, THF/H2O (1:1) r.t, 3 h (89%).
Based on these data, compound 7 was not further investigated, compound 14 being the most potent hCA XII inhibitor (Ki for hCA
and a structure-activity relationship (SAR) study was undertaken XII 3.8 nM), showing also a good SR with respect to hCA II
on 6. In particular, different substitution patterns on the pendant (SR 18.1).
2-phenyl ring at 5-position of benzimidazole were investigated
Specifically, the introduction of a second polar group (OH or
(compounds 9–17). Deletion of the 40-hydroxy substituent (9), as COOH) at the ortho position to the phenol ring positively affected
well as its replacement with methoxycarbonyl (10) or carboxy (11) the interaction with isoform XII (compounds 12 and 14), while the
groups, produces good but unselective inhibitors; in fact, a mod- presence of the methoxycarbonyl group (compound 13) resulted
est gain (9, 11) or a subsistence (10) in activity toward the isoform in a minor increase in hCA XII inhibition with respect to parent
I and a general decrease in inhibitory potency for hCA II, IX, and compound 6.
XII with respect to the lead 6 were observed, suggesting a precise
Concerning the IX isoform, an exactly opposite trend can be
role played by the hydroxy group in the interaction with the observed for compounds 12–14. Decoration of the 2-phenyl moi-
enzyme. This is confirmed by the inhibitory activities showed by ety with a 40-hydroxy and 30-methoxycarbonyl groups gives a
compounds 12–14, where the introduction of an o-substituent to highly effective hCA IX inhibitor (13), which shows low nanomolar
the p-hydroxy group on the 2-phenyl ring resulted in a moderate Ki (2.2 nM), with a relevant gain in activity compared to the refer-
increase in activity toward the two cytosolic CA evaluated, and a ence 1 (Ki 25 nM), and good SR vs hCA II. Differently, compounds
more considerable improvement for the isoform XII, with 12 and 14, featuring 30,40-dihydroxy and 30-carboxy-40-hydroxy

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1705
Scheme 4. (a) SOCl2, reflux, 2 h, then tert-butylamine, NEt3, THF, 0 ꢄC to r.t., 18 h (84%); (b) ammonium formate, Pd/C 10%, MeOH, reflux, 2 h (94%); (c) 25e, NaHSO3,
dry DMF, 80 ꢄC, 18h (72%); (d) DCM/TFA (1:1), r.t. 24 h, then LiOH, THF/H2O (1:1) r.t.,4 h (91%).
Table 1. Inhibition data of human CA I, II, IX, and XII isoforms with compounds 6–19 and the standard sulfonamide inhibitor AAZ (1) by a stopped-flow CO2 hydrase
assay.
Ki (nM)a
SRb
SRb
Compound
hCA I
250
237.9
hCA II
12
101.2
hCA IX
25
hCA XII
5.7
IX/II
XII/II
1 (AAZ)
6
0.48
5.8
2.1
2.3
17.4
44.1
7
8
442.1
213.6
91.9
47.8
73.9
14.4
63.8
1.2
3.3
1.4
4.9
9.8
9
92.8
208.2
97.6
104.1
185.4
133.2
79.0
47.6
29.6
64.3
41.6
78.9
72.2
57.0
8.5
2.2
6.3
2.1
1.9
1.3
2.6
2.3
5.8
10
11
12
169.3
13
14
125.5
95.6
50.8
68.6
2.2
22.3
3.8
23.1
2.0
2.3
34.2
18.1
(continued)

1706
C. MILITE ET AL.
Table 1. Continued.
Ki (nM)a
hCA IX
SRb
SRb
Compound
hCA I
hCA II
8921.1
hCA XII
813.1
IX/II
XII/II
15
>10000
390.5
665.1
5.9
–
–
16
17
>10000
7582.9
52.7
464.2
7.9
–
–
427.3
8.9
6.7
18
19
441.4
24.8
7.6
4.2
3.3
5.9
>10000
>10000
>10000
>10000
–
–
aMean from 3 different assays, by a stopped-flow technique (errors were in the range of 5–10% of the reported values).
bSR: Selectivity Ratio.
Figure 2. (a) 13/hCA IX (PDB 5FL4) theoretical complex as calculated by docking simulations. The protein is shown as cyan ribbons and sticks while the ligand as sal-
mon sticks. Critical residues are labeled. H-bonds are depicted as red dashed lines while coordination bonds as green dashed lines. (b) 13 hCA IX theoretical binding
pose within the hCA XII (PDB 5MSA) X-ray structure29. The protein is shown as light blue ribbons and sticks while the ligand as salmon sticks. Critical residues are
labeled. H-bonds are depicted as red dashed lines while coordination bonds as green dashed lines. (c) 13 hCA IX theoretical binding pose within the hCA I (PDB 6F3B)
structure. The protein is shown as green ribbons and its molecular surface in transparent gray. The ligand is represented as salmon sticks. (d) 13 hCA IX theoretical
binding pose within the hCA II (PDB 3K34) structure. The protein is shown as pink ribbons and its molecular surface in transparent gray. The ligand is represented as
salmon sticks. The images were rendered using the UCSF Chimera software30.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1707
Figure 3. (a) 14/hCA IX (PDB 5FL4) theoretical complex as calculated by docking simulations. The protein is shown as cyan ribbons and sticks while the ligand as
orange sticks. Critical residues are labeled. H-bonds are depicted as red dashed lines while coordination bonds as green dashed lines. (b) 14 hCA IX theoretical binding
pose within the hCA XII (PDB 5MSA) X-ray structure. The protein is shown as light blue ribbons and sticks while the ligand as orange sticks. Critical residues are
labeled. H-bonds are depicted as red dashed lines while coordination bonds as green dashed lines. (c) 14 hCA IX theoretical binding pose within the hCA I (PDB 6F3B)
X-ray structure. The protein is shown as green ribbons and its molecular surface as transparent gray. The ligand is shown as orange sticks. (d) 14 hCA IX docked bind-
ing pose within the hCA II (PDB 3K34) structure. The protein is shown as pink ribbons and its molecular surface in transparent gray. The ligand is depicted as orange
sticks. The images were rendered using the UCSF Chimera software30.
substituents, although being rather effective hCA IX inhibitors (Ki phenyl ring abolished the selectivity for hCA IX and XII isoforms,
for hCA IX 41.6 nM and 34.2 nM for 12 and 14, respectively), show that was observed for compounds 13 and 14, respectively.
Noteworthy, compounds 17 and 18 are better inhibitors than
the phenol derivative 8, proving the role of the ortho-carboxy-
methyl phenol and the ortho-carboxy phenol rings as proper scaf-
folds in the development of potent and selective hCA IX and hCA
XII inhibitors.
a slight decrease in activity with respect to reference compound 6
(Ki for hCA IX 17.4 nM).
These data suggested the ortho-carboxy phenol ring of com-
pound 14 and the ortho-carboxymethyl phenol ring of compound
13 as proper scaffolds for activity and selectivity for hCA XII and
hCA IX, respectively. For these derivatives, further SARs were
investigated.
First, we explored structural modifications of the 5-sulfonamide
moiety, including the insertion of a small alkyl group at the nitro-
gen atom to produce secondary sulfonamides (compounds 15
and 16), and replacement of the sulfonamide with a carboxamide
(19). The obtained compounds proved to be scarcely active or
completely inactive inhibitors of all the CA isoforms tested (Kis
varying from 390.5 nM to micromolar values), strongly supporting
the crucial role played by the primary sulfonamide group in the
interaction with the enzyme.
Molecular docking studies
To clarify the reasons for the activities displayed by the newly
designed compounds, molecular docking studies were attained.
Docking calculations were performed using a protocol already suc-
cessfully applied in our previous work on CA inhibitors39. Namely,
AutoDock4.2 (AD4)30,37 was employed together with the
AutoDock4(Zn) forcefield31, which was specifically designed to
accurately predict the binding interactions of ligands docking to
zinc metalloproteins.
Finally, we explored the effect of the combination of the ethyl
linker between the benzimidazole and the 2-substituted phenol
moieties (compounds 17 and 18 in Table 1). The potency for the
isoform hCA I decreased, while hCA II was slightly more (18) or
equally inhibited (17) with respect to related compounds 14 and
13, respectively. Both 17 and 18 derivatives resulted to be potent
hCA IX and hCA XII inhibitors, showing low nanomolar Ki values
(17, Ki for hCA IX 5.9 nM, Ki for hCA XII 7.9 nM; 18, Ki for hCA IX
Ligands 13, 14 and 17 were selected for the in silico experi-
ments as representative of the whole set. These compounds were
first docked in the active site of hCA IX. For the latter, a high-reso-
lution X-ray crystal structure bound to a small molecule inhibitor
(PDB code 5FL4)32 was chosen. According to our theoretical model
(Figures 2(A), 3(A) and 4(A)), in the three inspected ligands, the
negative nitrogen of the sulfonamide group chelates the zinc ion
of the active site. The sulfonamide also engages an H-bond with
7.6 nM, Ki for hCA XII 4.2 nM). However, the presence of the ethyl- the backbone of T200. Furthermore, the benzimidazole nitrogen is
ene linker between the benzimidazole scaffold and the side in a potential H-bond accepting position with Q92 side chain. The

1708
C. MILITE ET AL.
Figure 4. (a) 17/hCA IX (PDB 5FL4) theoretical complex as calculated by docking simulations. The protein is shown as cyan ribbons and sticks while the ligand as yel-
low sticks. Critical residues are labeled. H-bonds are depicted as red dashed lines while coordination bonds as green dashed lines. (b) 17 hCA IX theoretical binding
pose within the hCA XII (PDB 5MSA) X-ray structure. The protein is shown as light blue ribbons and sticks while the ligand as yellow sticks. Critical residues are
labeled. H-bonds are depicted as red dashed lines while coordination bonds as green dashed lines. (c) 17 hCA IX theoretical binding pose within the hCA I (PDB 6F3B)
X-ray structure. The protein is shown as green ribbons and its molecular surface as transparent gray. The ligand is shown as yellow sticks. (d) 17 hCA IX docked bind-
ing pose within the hCA II (PDB 3K34) structure. The protein is shown as pink ribbons and its molecular surface in transparent gray. The ligand is depicted as yellow
sticks. The images were rendered using the UCSF Chimera software30.
benzimidazole core also engages in contacts with the V121, L199, hCA IX, conducive of potent enzyme inhibition, is unlikely to be
and T201 sidechains (Figure 2(A), Figure 3(A) and Figure 4(A)).
Notably, it would appear that the pendant 2-phenyl ring, in the
three ligands, points towards what has been defined as a
“selectivity hot spot” in CAs40 (Figures 2(A), 3(A) and 4(A)): a high-
variability region in CAs binding site, that can be exploited for the
rational design of selective compounds among different CAs.
Here, ligand 13 phenyl ring and its 30-methoxycarbonyl moiety
are able to establish favorable contacts with the lipophilic side-
chains of L91 and V130 (Figure 2(A)). On the other hand, the 40-
hydroxy group is pointing outside of the binding site, probably
establishing a network of stabilizing H-bonding interactions with
the solvent water molecules (Figure 2(A)). It can be argued that
this accounts for the higher potency displayed by compounds fea-
turing the 40-hydroxy group. The same holds true for compound
14. As for ligand 17, while the benzimidazole core binding mode
is conserved, the ethyl linker engenders greater flexibility which
allows the side phenyl ring to expand further into the hotspot
gorge. This allows enhancing the positive contacts with L91 and
V130. Moreover, an additional H-bond between the 30-methoxy-
carbonyl group and Q71 is formed (Figure 4(A)).
confirmed for hCA I and hCA II due to major steric clashes
(Figures 2(C,D), 3(C,D), and 4(C,D)). Indeed, their binding sites in
the hot spot region feature bulky substituents that would unfavor-
ably affect the binding mode of the compounds, especially in the
case of ligand 17 (Figure 4(C,D)).
Conversely, hCA XII and hCA IX binding sites share a higher
degree of homology. As such, the binding poses found for 13, 14
and 17 in hCA IX also fit in hCA XII (Figures 2(B) and 3(B)). Still,
few key differences in the hot spot region can be found.
Specifically, some hydrophobic residues in hCA IX are replaced by
polar ones (L91, L123, V130 in CA IX become T88, Y121, S130 in
hCA XII, respectively). Importantly, in hCA XII the positive K69
takes the place of Q71 in hCA IX. It is possible to infer that the
more hydrophilic and positively charged binding site of hCA XII
provides a better fit for compounds with a 30-carboxyl group on
the pendant 2-phenyl ring (see compound 14, Figure 3(B)).
Instead, compounds bearing the 30-methoxycarbonyl moiety can
interact more favorably with the lipophilic and neutral hot spot
region of hCA IX. Purportedly, the presence of the ethyl linker
grants the possibility for the ligand to maximize the favorable
interactions in hCA IX and hCA XII, with both substitution patterns
on the 2-phenyl ring, the 30-carboxyl (18) or the 30-methoxycar-
bonyl (17) moiety. On the other hand, the same ethyl linker, by
enhancing the ligand flexibility, should also allow for a better fit
into the hCA I and hCA II isoform structures, thereby negatively
With the aim of rationalizing the selectivity of the compounds,
the crystal structures of hCA I (PDB 6F3B)34, hCA II (PDB 3K34)35,
and hCA XII (PDB 5MSA)36 were downloaded and their binding
sites analyzed. To ascertain how the predicted docked poses of
ligands 13, 14 and 17 in the hCA IX active site would fit in the
other CAs, the 3 D structures of the enzymes were superimposed.
This analysis revealed that the recognition pattern achieved for impacting on the ligand selectivity profile (Figures 2, 3 and 4).

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1709
Conclusions
References
Several Schiff bases and secondary amines incorporating aromatic
sulfonamide moieties in their structure has been extensively
studied as CAIs. Starting from these classes of compounds and
according to the frozen analog approach, we designed a series of
derivatives featuring the 2-substituted-benzimidazole-6-sulfona-
mide scaffold, a chemical template only scarcely exploited in the
CAIs’ medicinal chemistry field. A library of 14 derivatives was syn-
thesized and tested for their enzyme inhibitory activity against the
physiologically relevant human CA I, II, IX, and XII isoforms.
Computational studies were attained to rationalize the SAR in
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Of note, the identification of a number of newly synthesized
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Elisabetta Barresi
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